Modified compstatin with peptide backbone and c-terminal modifications

ABSTRACT

Compounds comprising peptides capable of binding C3 protein and inhibiting complement activation are disclosed. These compounds display greatly improved complement activation-inhibitory activity as compared with currently available compounds. The compounds comprise compstatin analogs having a constrained backbone at position 8 (glycine) and, optionally, specific substitutions for threonine at position 13.

GOVERNMENT SUPPORT

Pursuant to 35 U.S.C. §202(c), it is acknowledged that the United Statesgovernment may have certain rights in the invention described herein,which was made in part with funds from the National Institutes of Healthunder Grant No. GM 62134.

FIELD OF THE INVENTION

This invention relates to activation of the complement cascade in thebody. In particular, this invention provides peptides andpeptidomimetics capable of binding the C3 protein and inhibitingcomplement activation.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety.

The human complement system is a powerful player in the defense againstpathogenic organisms and the mediation of immune responses. Complementcan be activated through three different pathways: the classical,lectin, and alternative pathways. The major activation event that isshared by all three pathways is the proteolytic cleavage of the centralprotein of the complement system, C3, into its activation products C3aand C3b by C3 convertases. Generation of these fragments leads to theopsonization of pathogenic cells by C3b and iC3b, a process that rendersthem susceptible to phagocytosis or clearance, and to the activation ofimmune cells through an interaction with complement receptors(Markiewski & Lambris, 2007, Am J Pathol 171: 715-727). Deposition ofC3b on target cells also induces the formation of new convertasecomplexes and thereby initiates a self-amplification loop.

An ensemble of plasma and cell surface-bound proteins carefullyregulates complement activation to prevent host cells from self-attackby the complement cascade. However, excessive activation orinappropriate regulation of complement can lead to a number ofpathologic conditions, ranging from autoimmune to inflammatory diseases(Holers, 2003, Clin Immunol 107: 140-51; Markiewski & Lambris, 2007,supra; Ricklin & Lambris, 2007, Nat Biotechnol 25: 1265-75; Sahu et al.,2000, J Immunol 165: 2491-9). The development of therapeutic complementinhibitors is therefore highly desirable. In this context, C3 and C3bhave emerged as promising targets because their central role in thecascade allows for the simultaneous inhibition of the initiation,amplification, and downstream activation of complement (Ricklin &Lambris, 2007, supra).

Compstatin was the first non-host-derived complement inhibitor that wasshown to be capable of blocking all three activation pathways (Sahu etal., 1996, J Immunol 157: 884-91; U.S. Pat. No. 6,319,897). This cyclictridecapeptide binds to both C3 and C3b and prevents the cleavage ofnative C3 by the C3 convertases. Its high inhibitory efficacy wasconfirmed by a series of studies using experimental models that pointedto its potential as a therapeutic agent (Fiane et al., 1999a,Xenotransplantation 6: 52-65; Fiane et al., 1999b, Transplant Proc31:934-935; Nilsson et al., 1998 Blood 92: 1661-1667; Ricklin & Lambris,2008, Adv Exp Med Biol 632: 273-292; Schmidt et al., 2003, J BiomedMater Res A 66: 491-499; Soulika et al., 2000, Clin Immunol 96:212-221). Progressive optimization of compstatin has yielded analogswith improved activity (Ricklin & Lambris, 2008, supra; WO2004/026328;WO2007/062249). One of these analogs is currently being tested inclinical trials for the treatment of age-related macular degeneration(AMD), the leading cause of blindness in elderly patients inindustrialized nations (Coleman et al., 2008, Lancet 372: 1835-1845;Ricklin & Lambris, 2008, supra). In view of its therapeutic potential inAMD and other diseases, further optimization of compstatin to achieve aneven greater efficacy is of considerable importance.

Earlier structure-activity studies have identified the cyclic nature ofthe compstatin peptide and the presence of both a β-turn and hydrophobiccluster as key features of the molecule (Morikis et al., 1998, ProteinSci 7: 619-627; W099/13899; Morikis et al., 2002, J Biol Chem277:14942-14953; Ricklin & Lambris, 2008, supra). Hydrophobic residuesat positions 4 and 7 were found to be of particular importance, andtheir modification with unnatural amino acids generated an analog with264-fold improved activity over the original compstatin peptide(Katragadda et al., 2006, J Med Chem 49: 4616-4622; WO2007/062249).

While previous optimization steps have been based on combinatorialscreening studies, solution structures, and computational models (Chiuet al., 2008, Chem Biol Drug Des 72: 249-256; Mulakala et al., 2007,Bioorg Med Chem 15: 1638-1644; Ricklin & Lambris, 2008, supra), therecent publication of a co-crystal structure of compstatin complexedwith the complement fragment C3c (Janssen et al., 2007, J Biol Chem 282:29241-29247; WO2008/153963) represents an important milestone forinitiating rational optimization. The crystal structure revealed ashallow binding site at the interface of macroglobulin (MG) domains 4and 5 of C3c and showed that 9 of the 13 amino acids were directlyinvolved in the binding, either through hydrogen bonds or hydrophobiceffects. As compared to the structure of the compstatin peptide insolution (Morikis et al., 1998, supra), the bound form of compstatinexperienced a conformational change, with a shift in the location of theβ-turn from residues 5-8 to 8-11 (Janssen et al., 2007, supra;WO2008/153963).

In view of the foregoing, it is clear that the development of modifiedcompstatin peptides or mimetics with even greater activity wouldconstitute a significant advance in the art.

SUMMARY OF THE INVENTION

The present invention provides analogs and mimetics of thecomplement-inhibiting peptide, compstatin, ICVVQDWGHHRCT (cyclicC2-C12); SEQ ID NO:1), which have improved complement-inhibitingactivity as compared to compstatin.

One aspect of the invention features a compound comprising a modifiedcompstatin peptide (ICVVQDWGHHRCT (cyclic C2-C12); SEQ ID NO:1) oranalog thereof, in which the Gly at position 8 is modified to constrainthe backbone conformation of the peptide at that location. In oneembodiment, the backbone is constrained by replacing the Gly withN-methyl Gly. The peptide may be further modified by one or more of:replacement of His at position 9 with Ala; replacement of Val atposition 4 with Trp or an analog of Trp; replacement of Trp at position7 with an analog of Trp; acetylation of the N-terminal residue; andreplacement of Thr at position 13 with Ile, Leu, Nle, N-methyl Thr orN-methyl Ile. In particular embodiments, the analog of Trp at position 4is 1-methyl Trp or 1-formyl Trp and the analog of Trp at position 7, ifpresent, is a halogenated Trp.

Certain embodiments feature a compstatin analog comprising a peptidehaving a sequence of SEQ ID NO:2, which is:

Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa3-Gly-Xaa4-His-Arg-Cys-Xaa5 (cyclic C2-C12)in which Gly at position 8 is modified to constrain the backboneconformation of the peptide at that location, and wherein:

-   Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide    comprising Gly-Ile;-   Xaa2 is Trp or an analog of Trp, wherein the analog of Trp has    increased hydrophobic character as compared with Trp;-   Xaa3 is Trp, or an analog of Trp comprising a chemical modification    to its indole ring wherein the chemical modification increases the    hydrogen bond potential of the indole ring;-   Xaa4 is His, Ala, Phe or Trp; and-   Xaa5 is Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile, wherein a    carboxy terminal —OH of any of the Thr, Ile, Leu, Nle, N-methyl Thr    or N-methyl Ile optionally is replaced by —NH₂.

In certain embodiments, Xaa2 participates in a nonpolar interaction withC3. In other embodiments, Xaa3 participates in a hydrogen bond with C3.In various embodiments, the analog of Trp of Xaa2 is a halogenatedtrpytophan, such as 5-fluoro-1-tryptophan or 6-fluoro-1-tryptophan. Inother embodiments, the Trp analog at Xaa2 comprises a lower alkoxy orlower alkyl substituent at the 5 position, e.g., 5-methoxytryptophan or5-methyltryptophan. In other embodiments, the Trp analog at Xaa 2comprises a lower alkyl or a lower alkenoyl substituent at the 1position, with exemplary embodiments comprising 1-methyltryptophan or1-formyltryptophan. In other embodiments, the analog of Trp of Xaa3 is ahalogenated tryptophan such as 5-fluoro-1-tryptophan or6-fluoro-1-tryptophan. In particular embodiments, Xaa2 is1-methyltryptophan or 1-formyltryptophan and Xaa3 optionally comprises5-fluoro-1-tryptophan.

In certain embodiments, the Gly at position 8 is N-methylated, and Xaa1is Ac-Ile, Xaa2 is 1-methyl-Trp or 1-formyl-Trp, Xaa3 is Trp, Xaa4 isAla, and Xaa5 is Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile. Inparticular, Xaa5 may be Ile, N-methyl Thr or N-methyl Ile. Inparticular, the compstatin analog comprises any one of SEQ ID NOS: 5, 7,8, 9, 10 or 11.

In some embodiments, the compound comprises a peptide produced byexpression of a polynucleotide encoding the peptide. In otherembodiments, the compound is produced at least in part by peptidesynthesis. A combination of synthetic methods can also be used.

Another aspect of the invention features a compound of any of thepreceding claims, further comprising an additional component thatextends the in vivo retention of the compound. The additional componentis polyethylene glycol (PEG) in one embodiment. The additional componentis an albumin binding small molecule in another embodiment. In anotherembodiment, the additional component is an albumin binding peptide. Thealbumin binding peptide may comprise the sequence RLIEDICLPRWGCLWEDD(SEQ ID NO: 14). Particular embodiments comprise any one of SEQ ID NOS:5, 7, 8, 9, 10 or 11 linked to the albumin binding peptide. Optionally,the compound and the albumin binding peptide are separated by a spacer.The spacer can be a polyethylene glycol (PEG) molecule, such as mini-PEGor mini-PEG 3.

Another aspect of the invention features compound that inhibitscomplement activation, comprising a non-peptide or partial peptidemimetic of any one of SEQ ID NOS: 5, 7, 8, 9, 10 or 11, wherein thecompound binds C3 and inhibits complement activation with at least500-fold greater activity than does a peptide comprising SEQ ID NO:1under equivalent assay conditions.

The compstatin analogs, conjugates and mimetics of the invention are ofpractical utility for any purpose for which compstatin itself isutilized, as known in the art and described in greater detail herein.Certain of these uses involve the formulation of the compounds intopharmaceutical compositions for administration to a patient. Suchformulations may comprise pharmaceutically acceptable salts of thecompounds, as well as one or more pharmaceutically acceptable diluents,carriers excipients, and the like, as would be within the purview of theskilled artisan.

Various features and advantages of the present invention will beunderstood by reference to the detailed description, drawings andexamples that follow.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions:

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definition provided herein.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, in some embodiments ±5%, in someembodiments ±1%, and in some embodiments ±0.1% from the specified value,as such variations are appropriate to make and used the disclosedcompounds and compositions.

The term “compstatin” as used herein refers to a peptide comprising SEQID NO:1, ICVVQDWGHHRCT (cyclic C2-C12). The term “compstatin analog”refers to a modified compstatin comprising substitutions of natural andunnatural amino acids, or amino acid analogs, as well as modificationswithin or between various amino acids, as described in greater detailherein, and as known in the art. When referring to the locationparticular amino acids or analogs within compstatin or compstatinanalogs, those locations are sometimes referred to as “positions” withinthe peptide, with the positions numbered from 1 (Ile in compstatin) to13 (Thr in compstatin). For example, the Gly residue occupies “position8.”

The terms “pharmaceutically active” and “biologically active” refer tothe ability of the compounds of the invention to bind C3 or fragmentsthereof and inhibit complement activation. This biological activity maybe measured by one or more of several art-recognized assays, asdescribed in greater detail herein.

As used herein, “alkyl” refers to an optionally substituted saturatedstraight, branched, or cyclic hydrocarbon having from about 1 to about10 carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein), with from about 1 to about 7carbon atoms being preferred. Alkyl groups include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,n-pentyl, cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl,cyclohexyl, cyclooctyl, adamantyl, 3-methylpentyl, 2,2-dimethylbutyl,and 2,3-dimethylbutyl. The term “lower alkyl” refers to an optionallysubstituted saturated straight, branched, or cyclic hydrocarbon havingfrom about 1 to about 5 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein).Lower alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl,isopentyl and neopentyl.

As used herein, “halo” refers to F, Cl, Br or I.

As used herein, “alkanoyl”, which may be used interchangeably with“acyl”, refers to an optionally substituted a straight or branchedaliphatic acylic residue having from about 1 to about 10 carbon atoms(and all combinations and subcombinations of ranges and specific numbersof carbon atoms therein), with from about 1 to about 7 carbon atomsbeing preferred. Alkanoyl groups include, but are not limited to,formyl, acetyl, propionyl, butyryl, isobutyryl pentanoyl, isopentanoyl,2-methyl-butyryl, 2,2-dimethylpropionyl, hexanoyl, heptanoyl, octanoyl,and the like. The term “lower alkanoyl” refers to an optionallysubstituted straight or branched aliphatic acylic residue having fromabout 1 to about 5 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein.Lower alkanoyl groups include, but are not limited to, formyl, acetyl,n-propionyl, iso-propionyl, butyryl, iso-butyryl, pentanoyl,iso-pentanoyl, and the like.

As used herein, “aryl” refers to an optionally substituted, mono- orbicyclic aromatic ring system having from about 5 to about 14 carbonatoms (and all combinations and subcombinations of ranges and specificnumbers of carbon atoms therein), with from about 6 to about 10 carbonsbeing preferred. Non-limiting examples include, for example, phenyl andnaphthyl.

As used herein, “aralkyl” refers to alkyl radicals bearing an arylsubstituent and have from about 6 to about 20 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 6 to about 12 carbon atoms beingpreferred. Aralkyl groups can be optionally substituted. Non-limitingexamples include, for example, benzyl, naphthylmethyl, diphenylmethyl,triphenylmethyl, phenylethyl, and diphenylethyl.

As used herein, the terms “alkoxy” and “alkoxyl” refer to an optionallysubstituted alkyl-O— group wherein alkyl is as previously defined.Exemplary alkoxy and alkoxyl groups include methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, and heptoxy, among others.

As used herein, “carboxy” refers to a —C(═O)OH group.

As used herein, “alkoxycarbonyl” refers to a —C(═O)O-alkyl group, wherealkyl is as previously defined.

As used herein, “aroyl” refers to a —C(═O)-aryl group, wherein aryl isas previously defined. Exemplary aroyl groups include benzoyl andnaphthoyl.

Typically, substituted chemical moieties include one or moresubstituents that replace hydrogen at selected locations on a molecule.Exemplary substituents include, for example, halo, alkyl, cycloalkyl,aralkyl, aryl, sulfhydryl, hydroxyl (—OH), alkoxyl, cyano (—CN),carboxyl (—COOH), acyl (alkanoyl: —C(═O)R); —C(═O)O-alkyl, aminocarbonyl(—C(═O)NH₂), —N-substituted aminocarbonyl (—C(═O)NHR″), CF₃, CF₂CF₃, andthe like. In relation to the aforementioned substituents, each moiety R″can be, independently, any of H, alkyl, cycloalkyl, aryl, or aralkyl,for example.

As used herein, “L-amino acid” refers to any of the naturally occurringlevorotatory alpha-amino acids normally present in proteins or the alkylesters of those alpha-amino acids. The term D-amino acid” refers todextrorotatory alpha-amino acids. Unless specified otherwise, all aminoacids referred to herein are L-amino acids.

“Hydrophobic” or “nonpolar” are used synonymously herein, and refer toany inter- or intra-molecular interaction not characterized by a dipole.

“PEGylation” refers to the reaction in which at least one polyethyleneglycol (PEG) moiety, regardless of size, is chemically attached to aprotein or peptide to form a PEG-peptide conjugate. “PEGylated meansthat at least one PEG moiety, regardless of size, is chemically attachedto a peptide or protein. The term PEG is generally accompanied by anumeric suffix that indicates the approximate average molecular weightof the PEG polymers; for example, PEG-8,000 refers to polyethyleneglycol having an average molecular weight of about 8,000.

As used herein, “pharmaceutically-acceptable salts” refers toderivatives of the disclosed compounds wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically-acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Thus, the term “acid addition salt” refers to the correspondingsalt derivative of a parent compound that has been prepared by theaddition of an acid. The pharmaceutically-acceptable salts include theconventional salts or the quaternary ammonium salts of the parentcompound formed, for example, from inorganic or organic acids. Forexample, such conventional salts include, but are not limited to, thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like. Certain acidic orbasic compounds of the present invention may exist as zwitterions. Allforms of the compounds, including free acid, free base, and zwitterions,are contemplated to be within the scope of the present invention.

Description:

In accordance with the present invention, information about thebiological and physico-chemical characteristics of compstatin binding toC3 have been employed to design modified compstatin peptides withsignificantly improved activity compared to the parent compstatinpeptide. In some embodiments, the analogs have at least 300-fold greateractivity than does compstatin. In other embodiments, the analogs have350-, 400-, 450-, 500-, 550-, 600-fold or greater activity than doescompstatin, as compared utilizing the assays described in the examples.

Compstatin analogs synthesized in accordance with previous approacheshave been shown to possess improved activity as compared with the parentpeptide, i.e., up to about 99-fold (Mallik, B. et al, 2005, supra;WO2004/026328), and up to about 264-fold (Katragadda et al., 2006,supra; WO2007/062249). The analogs produced in accordance with thepresent invention demonstrate improved activity via modification at aposition of compstatin heretofore not utilized, and can impart improvedactivity to compstatin or any currently described analog. The analogs ofthe present invention thus possess even greater activity than either theparent peptide or analogs thereof produced to date, as demonstrated byin vitro assays as shown in the figures and in the Examples herein.

The table below shows amino acid sequence and complement inhibitoryactivities of selected exemplary analogs with significantly improvedactivity over compstatin (Ic[CVVQDWGHHRC]T; SEQ ID NO:1). The selectedanalogs are referred to by specific modifications of designatedpositions (1-13) as compared to a potent compstatin analog(Ac-Ic[CV(^(1-Me)W)QDWGAHRC]T-NH₂, SEQ ID NO:4, also referred to aspeptide 14 in Example 1) which was described in WO2007/062249. Thepeptides of SEQ ID NOS: 5 and 7-11 (also referred to as peptides 15 and17-21 in Example 1) are representative of modifications made inaccordance with the present invention, resulting in significantly morepotent compstatin analogs.

Exemplary compstatin analogs, IC₅₀, and fold change in activity relativeto SEQ ID NO:4 (Ac-Ic[CV(^(1-Me)W)QDWGAHRC]T-NH₂), IC₅₀ of 206 nM):

SEQ ID Pept. IC₅₀ Fold NO: No. Xaa⁸ Xaa¹³ Sequence (nM) change 5 15 Sar*Thr Ac−Ic[CV(^(1−Me)W)QDW(^(N−Me)G)AHRC]T-NH₂ 159 1.30 7 17 Sar IleAc−Ic[CV(^(1−Me)W)QDW(^(N−Me)G)AHRC]I-NH₂ 92 2.24 8 18 Sar LeuAc−Ic[CV(^(1−Me)W)QDW(^(N−Me)G)AHRC]L-NH₂ 108 1.91 9 19 Sar NleAc−Ic[CV(^(1−Me)W)QDW(^(N−Me)G)AHRC](Nle)-NH₂ 109 1.90 10 20 Sar^((NMe))Thr  Ac−Ic[CV(^(1−Me)W)QDW(^(N−Me)G)AHRC](^(N−Me)T)-NH₂ 86 2.4011 21 Sar ^((NMe))Ile Ac−Ic[CV(^(1−Me)W)QDW(^(N−Me)G)AHRC](^(N−Me)I)-NH₂62 3.32 *Sar = N-Me Gly

One modification in accordance with the present invention comprisesconstraint of the peptide backbone at position 8 of the peptide. In aparticular embodiment, the backbone is constrained by replacing glycineat position 8 (Gly⁸) with N-methyl glycine. Reference is made toexemplary peptides 8 and 15 as discussed in Example 1.

Without intending to be bound or limited by theory, it is noted thatN-methylation can affect a peptide in several ways. First, the potentialhydrogen bond donor is replaced with a methyl group, which cannot form ahydrogen bond. Second, the N-methyl group is weakly electron-donatingwhich means it can slightly increase the basicity of the neighboringcarbonyl group. Third, the size of the N-methyl group could cause stericconstraint. Finally, the N-methylation can change the trans/cispopulation of the amide bond, thus changing local peptide conformationin a manner similar to a proline.

The activity increase of [Trp(Me)⁴Gly(N-Me)⁸Ala⁹]-Ac-compstatin (SEQ IDNO:5; peptide 15) is a noteworthy improvement as compared to thepreviously most active analog, [Trp(Me)⁴Gly⁸Ala⁹]-Ac-compstatin (SEQ IDNO:4; peptide 14). N-methylation of Gly⁸ likely improves targetrecognition and complex stability by reinforced bound-like β-turn,increased local backbone constraints and improved hydrophobicinteractions involving the side chain of Trp⁷.

In particular embodiments, the modification at position 8 issupplemented with an additional modification comprising replacing Thr atposition 13 with Ile, Leu, Nle (norleucine), N-methyl Thr or N-methylIle. Reference is made to exemplary peptides 16, 17, 18, 19, 20 and 21(SEQ ID NOS: 6, 7, 8, 9, 10 and 11) as discussed in Example 1. Again,without intending to be limited or bound by theory, replacement of Thrwith hydrophobic Ile was found to be beneficial. The similar effectsobserved for the two isomers of Ile (i.e., Leu and Nle) suggest thatphysicochemical and steric properties, rather than specific contacts,may be responsible for this improvement. However, a more distinctimprovement in affinity and activity was observed upon backboneN-methylation of both Thr¹³ and Ile¹³. While the observed improvementsmay have resulted from increased backbone restraints, and hence lowerconformational entropic penalties upon binding, it is also the case thatthe nature of the residue at position 13 can further influence theformation and stabilization of active conformations, either stericallyor via formation of intramolecular hydrophobic contacts.

The above-described modifications at position 8 and position 13 can becombined with other modifications of compstatin previously shown toimprove activity, to produce peptides with significantly improvedcomplement inhibiting activity. For example, acetylation of theN-terminus typically increases the complement-inhibiting activity ofcompstatin and its analogs. Accordingly, addition of an acyl group atthe amino terminus of the peptide, including but not limited toN-acetylation, is one preferred embodiment of the invention, ofparticular utility when the peptides are prepared synthetically.However, it is sometimes of advantage to prepare the peptides byexpression of a peptide-encoding nucleic acid molecule in a prokaryoticor eukaryotic expression system, or by in vitro transcription andtranslation. For these embodiments, the naturally-occurring N-terminusmay be utilized.

As another example, it is known that substitution of Ala for His atposition 9 improves activity of compstatin and is a preferredmodification of the peptides of the present invention as well. It hasalso been determined that substitution of Tyr for Val at position 4 canresult in a modest improvement in activity (Klepeis et al., 2003, J AmChem Soc 125: 8422-8423).

It was disclosed in WO2004/026328 and WO2007/0622249 that Trp andcertain Trp analogs at position 4, as well as certain Trp analogs atposition 7, especially combined with Ala at position 9, yields many-foldgreater activity than that of compstatin. These modifications are usedto advantage in the present invention as well.

In particular, peptides comprising 5-fluoro-l-tryptophan or either5-methoxy-, 5-methyl- or 1-methyl-tryptophan, or 1-formyl-tryptophan atposition 4 have been shown to possess 31-264-fold greater activity thandoes compstatin. Particularly preferred are 1-methyl and 1-formyltryptophan. It is believed that an indole ‘N’-mediated hydrogen bond isnot necessary at position 4 for the binding and activity of compstatin.The absence of this hydrogen bond or reduction of the polar character byreplacing hydrogen with lower alkyl, alkanoyl or indole nitrogen atposition 4 enhances the binding and activity of compstatin. Withoutintending to be limited to any particular theory or mechanism of action,it is believed that a hydrophobic interaction or effect at position 4strengthens the interaction of compstatin with C3. Accordingly,modifications of Trp at position 4 (e.g., altering the structure of theside chain according to methods well known in the art), or substitutionsat position 4 or position 7 of Trp analogs that maintain or enhance theaforementioned hydrophobic interaction are contemplated in the presentinvention as an advantageous modification in combination with themodifications at positions 8 and 13 as described above. Such analogs arewell known in the art and include, but are not limited to the analogsexemplified herein, as well as unsubstituted or alternativelysubstituted derivatives thereof. Examples of suitable analogs may befound by reference to the following publications, and many others:Beene, et al., 2002, Biochemistry 41: 10262-10269 (describing, interalia, singly- and multiply-halogenated Trp analogs); Babitzky &Yanofsky, 1995, J. Biol. Chem. 270: 12452-12456 (describing, inter alia,methylated and halogenated Trp and other Trp and indole analogs); andU.S. Pat. Nos. 6,214,790, 6,169,057, 5,776,970, 4,870,097, 4,576,750 and4,299,838. Trp analogs may be introduced into the compstatin peptide byin vitro or in vivo expression, or by peptide synthesis, as known in theart.

In certain embodiments, Trp at position 4 of compstatin is replaced withan analog comprising a 1-alkyl substituent, more particularly a loweralkyl (e.g., C₁-C₅) substiutent as defined above. These include, but arenot limited to, N(α) methyl tryptophan, N(α) formyl tryptophan and5-methyltryptophan. In other embodiments, Trp at position 4 ofcompstatin is replaced with an analog comprising a 1-alkanoylsubstituent, more particularly a lower alkanoyl (e.g., C₁-C₅)substituent as defined above. In addition to exemplified analogs, theseinclude but are not limited to 1-acetyl-L-tryptophan andL-β-homotryptophan.

It was disclosed in WO2007/0622249 that incorporation of5-fluoro-l-tryptophan at position 7 in compstatin increased enthalpy ofthe interaction between compstatin and C3, relative to wildtypecompstatin, whereas incorporation of 5-fluoro-tryptophan at position 4in compstatin decreased the enthalpy of this interaction. Accordingly,modifications of Tip at position 7, as described in WO2007/0622249, arecontemplated as useful modifications in combination with themodifications to positions 8 and 13 as described above.

The modified compstatin peptides of the present invention may beprepared by various synthetic methods of peptide synthesis viacondensation of one or more amino acid residues, in accordance withconventional peptide synthesis methods. For example, peptides aresynthesized according to standard solid-phase methodologies, such as maybe performed on an Applied Biosystems Model 431A peptide synthesizer(Applied Biosystems, Foster City, Calif.), according to manufacturer'sinstructions. Other methods of synthesizing peptides or peptidomimetics,either by solid phase methodologies or in liquid phase, are well knownto those skilled in the art. During the course of peptide synthesis,branched chain amino and carboxyl groups may be protected/deprotected asneeded, using commonly-known protecting groups. An example of a suitablepeptide synthetic method is set forth in Example 1. Modificationutilizing alternative protecting groups for peptides and peptidederivatives will be apparent to those of skill in the art.

Alternatively, certain peptides of the invention may be produced byexpression in a suitable prokaryotic or eukaryotic system. For example,a DNA construct may be inserted into a plasmid vector adapted forexpression in a bacterial cell (such as E. coli) or a yeast cell (suchas Saccharomyces cerevisiae), or into a baculovirus vector forexpression in an insect cell or a viral vector for expression in amammalian cell. Such vectors comprise the regulatory elements necessaryfor expression of the DNA in the host cell, positioned in such a manneras to permit expression of the DNA in the host cell. Such regulatoryelements required for expression include promoter sequences,transcription initiation sequences and, optionally, enhancer sequences.

The peptides can also be produced by expression of a nucleic acidmolecule in vitro or in vivo. A DNA construct encoding a concatemer ofthe peptides, the upper limit of the concatemer being dependent on theexpression system utilized, may be introduced into an in vivo expressionsystem. After the concatemer is produced, cleavage between theC-terminal Asn and the following N-terminal G is accomplished byexposure of the polypeptide to hydrazine.

The peptides produced by gene expression in a recombinant procaryotic oreucaryotic system may be purified according to methods known in the art.A combination of gene expression and synthetic methods may also beutilized to produce compstatin analogs. For example, an analog can beproduced by gene expression and thereafter subjected to one or morepost-translational synthetic processes, e.g., to modify the N- orC-terminus or to cyclize the molecule.

Advantageously, peptides that incorporate unnatural amino acids, e.g.,methylated amino acids, may be produced by in vivo expression in asuitable prokaryotic or eukaryotic system. For example, methods such asthose described by Katragadda & Lambris (2006, Protein Expression andPurification 47: 289-295) to introduce unnatural Trp analogs intocompstatin via expression in E. coli auxotrophs may be utilized tointroduce N-methylated or other unnatural amino acids at selectedpositions of compstatin.

The structure of compstatin is known in the art, and the structures ofthe foregoing analogs are determined by similar means. Once a particulardesired conformation of a short peptide has been ascertained, methodsfor designing a peptide or peptidomimetic to fit that conformation arewell known in the art. Of particular relevance to the present invention,the design of peptide analogs may be further refined by considering thecontribution of various side chains of amino acid residues, as discussedabove (i.e., for the effect of functional groups or for stericconsiderations).

It will be appreciated by those of skill in the art that a peptide mimicmay serve equally well as a peptide for the purpose of providing thespecific backbone conformation and side chain functionalities requiredfor binding to C3 and inhibiting complement activation. Accordingly, itis contemplated as being within the scope of the present invention toproduce C3-binding, complement-inhibiting compounds through the use ofeither naturally-occurring amino acids, amino acid derivatives, analogsor non-amino acid molecules capable of being joined to form theappropriate backbone conformation. A non-peptide analog, or an analogcomprising peptide and non-peptide components, is sometimes referred toherein as a “peptidomimetic” or “isosteric mimetic,” to designatesubstitutions or derivations of the peptides of the invention, whichpossess the same backbone conformational features and/or otherfunctionalities, so as to be sufficiently similar to the exemplifiedpeptides to inhibit complement activation.

The use of peptidomimetics for the development of high-affinity peptideanalogs is well known in the art (see, e.g., Vagner et al., 2008, Curr.Opin. Chem. Biol. 12: 292-296; Robinson et al., 2008, Drug Disc. Today13: 944-951) Assuming rotational constraints similar to those of aminoacid residues within a peptide, analogs comprising non-amino acidmoieties may be analyzed, and their conformational motifs verified, byany variety of computational techniques that are well known in the art.

The modified compstatin peptides of the present invention can bemodified by the addition of polyethylene glycol (PEG) components to thepeptide. As is well known in the art, PEGylation can increase thehalf-life of therapeutic peptides and proteins in vivo. In oneembodiment, the PEG has an average molecular weight of about 1,000 toabout 50,000. In another embodiment, the PEG has an average molecularweight of about 1,000 to about 20,000. In another embodiment, the PEGhas an average molecular weight of about 1,000 to about 10,000. In anexemplary embodiment, the PEG has an average molecular weight of about5,000. The polyethylene glycol may be a branched or straight chain, andpreferably is a straight chain.

The compstatin analogs of the present invention can be covalently bondedto PEG via a linking group. Such methods are well known in the art.(Reviewed in Kozlowski A. et al. 2001, BioDrugs 15: 419-29; see also,Harris J M and Zalipsky S, eds. Poly(ethylene glycol), Chemistry andBiological Applications, ACS Symposium Series 680 (1997)). Non-limitingexamples of acceptable linking groups include an ester group, an amidegroup, an imide group, a carbamate group, a carboxyl group, a hydroxylgroup, a carbohydrate, a succinimide group (including withoutlimitation, succinimidyl succinate (SS), succinimidyl propionate (SPA),succinimidyl carboxymethylate (SCM), succinimidyl succinamide (SSA) andN-hydroxy succinimide (NHS)), an epoxide group, an oxycarbonylimidazolegroup (including without limitation, carbonyldimidazole (CDI)), a nitrophenyl group (including without limitation, nitrophenyl carbonate (NPC)or trichlorophenyl carbonate (TPC)), a trysylate group, an aldehydegroup, an isocyanate group, a vinylsulfone group, a tyrosine group, acysteine group, a histidine group or a primary amine. In certainembodiments, the linking group is a succinimide group. In oneembodiment, the linking group is NHS.

The compstatin analogs of the present invention can alternatively becoupled directly to PEG (i.e., without a linking group) through an aminogroup, a sulfhydral group, a hydroxyl group or a carboxyl group. In oneembodiment, PEG is coupled to a lysine residue added to the C-terminusof compstatin.

As an alternative to PEGylation, the in vivo clearance of peptides canalso be reduced by linking the peptides to certain other molecules orpeptides. For instance, certain albumin binding peptides display anunusually long half-life of 2.3 h when injected by intravenous bolusinto rabbits (Dennis et al., 2002, J Biol Chem. 277: 35035-35043). Apeptide of this type, fused to the anti-tissue factor Fab of D3H44enabled the Fab to bind albumin while retaining the ability of the Fabto bind tissue factor (Nguyen et al., 2006, Protein Eng Des Sel. 19:291-297.). This interaction with albumin resulted in significantlyreduced in vivo clearance and extended half-life in mice and rabbits,when compared with the wild-type D3H44 Fab, comparable with those seenfor PEGylated Fab molecules, immunoadhesins, and albumin fusions.WO2007/062249 describes a compstatin analog fused with analbumin-binding peptide (ABP) and reports that the fusion protein isactive in inhibiting complement activation. However, the synthesis waslengthy and the yield of fusion product was lower than desired. Example2 herein sets forth improved synthesis strategies utilizing an ABP aswell as an albumin-binding small molecule (ABM), and optionallyemploying a spacer between the components. Those procedures resulted inthe production of conjugates of ABP- and ABM-compstatin analogs capableof inhibiting complement activation and also exhibiting extended in vivosurvival. Indeed, the ABP was able to improve the half-life of acompstatin analog by 21 fold without significantly compromising itsinhibitory activity. Thus, such conjugates enable the systemicadministration of the inhibitor without infusion.

The complement activation-inhibiting activity of compstatin analogs,peptidomimetics and conjugates may be tested by a variety of assaysknown in the art. In one embodiment, the assay described in Example 1 isutilized. A non-exhaustive list of other assays is set forth in U.S.Pat. No. 6,319,897, WO99/13899, WO2004/026328 and WO2007/062249,including, but not limited to, (1) peptide binding to C3 and C3fragments; (2) various hemolytic assays; (3) measurement of C3convertase-mediated cleavage of C3; and (4) measurement of Factor Bcleavage by Factor D.

The peptides and peptidomimetics described herein are of practicalutility for any purpose for which compstatin itself is utilized, asknown in the art. Such uses include, but are not limited to: (1)inhibiting complement activation in the serum, tissues or organs of apatient (human or animal), which can facilitate treatment of certaindiseases or conditions, including but not limited to, age-relatedmacular degeneration, rheumatoid arthritis, spinal cord injury,Parkinson's disease, Alzheimer's disease, cancer, and respiratorydisorders such as asthma, chronic obstructive pulmonary disease (COPD),allergic inflammation, emphysema, bronchitis, bronchiecstasis, cycticfibrosis, tuberculosis, pneumonia, respiratory distress syndrome(RDS-neonatal and adult), rhinitis and sinusitis; (2) inhibitingcomplement activation that occurs during cell or organ transplantation,or in the use of artificial organs or implants (e.g., by coating orotherwise treating the cells, organs, artificial organs or implants witha peptide of the invention); (3) inhibiting complement activation thatoccurs during extracorporeal shunting of physiological fluids (blood,urine) (e.g., by coating the tubing through which the fluids are shuntedwith a peptide of the invention); and (4) in screening of small moleculelibraries to identify other inhibitors of compstatin activation (e.g.,liquid- or solid-phase high-throughput assays designed to measure theability of a test compound to compete with a compstatin analog forbinding with C3 or a C3 fragment).

To implement one or more of the utilities mentioned above, anotheraspect of the invention features pharmaceutical compositions comprisingthe compstatin analogs or conjugates described and exemplified herein.Such a pharmaceutical composition may consist of the active ingredientalone, in a form suitable for administration to a subject, or thepharmaceutical composition may comprise the active ingredient and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

The formulations of the pharmaceutical compositions may be prepared byany method known or hereafter developed in the art of pharmacology. Ingeneral, such preparatory methods include the step of bringing theactive ingredient into association with a carrier or one or more otheraccessory ingredients, and then, if necessary or desirable, shaping orpackaging the product into a desired single-or multi-does unit.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which a complement inhibitor may be combinedand which, following the combination, can be used to administer thecomplement inhibitor to a mammal.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

EXAMPLE 1

A mono-N^(β)-methylation scan was performed on [Tyr⁴Ala⁹]-Ac-compstatin(Ac-Ic[CVYQDWGAHRC]T-NH₂; SEQ ID NO:3). Based on the assay results ofthese analogs, selective N-methylation and substitution at position 13was performed on [Trp(Me)⁴Ala⁹]-Ac-compstatin(Ac-Ic[CV(^(1-Me)W)QDWGAHRC]T-NH₂; SEQ ID NO:4). Selected analogs werefurther characterized using surface plasmon resonance (SPR) andisothermal titration calorimetry (ITC). Molecular dynamics (MD)simulations were also performed to investigate possible mechanisms forthe observed increase in affinity.

Materials and Methods:

Abbreviations. Ac, acetyl group; Acm, acetamidomethyl; Boc,tert-butoxycarbonyl; CHARMM, Chemistry at Harvard MacromolecularMechanics; DCM, dichloromethane; DIC, 1,3-diisopropylcarbodiimide;DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethyl-formamide; ELISA,enzyme-linked immunosorbent assay; ESI, electrospray ionization; Fmoc,9-fluorenylmethoxycarbonyl; HOAt, 1-hydroxy-7-aza-benzotriazole; ITC,isothermal titration calorimetry; MALDI, matrix-assisted laserdesorption ionization; MBHA, 4-methylbenz-hydrylamine; MOE, molecularoperating environment; NAMD, nanoscale molecular dynamics; Nle,L-norleucine; NMP, N-methylpyrrolidinone; RMSD, root mean squaredeviation; SPR, surface plasmon resonance; TIPS, triisopropylsilane;Trt, trityl.

Chemicals. Low-loading Rink amide MBHA resin and the followingFmoc-amino acids were obtained from Novabiochem (San Diego, Calif.):Ile, Cys(Acm), Val, Tyr(tBu), Gln(Trt), Asp(OtBu), Trp(Boc), Gly, Sar,Ala, MeAla, His(Trt), Arg(Pmc), MeIle, Nle, Phe, and Thr(tBu). DIC andFmoc-Trp(Me)-OH were purchased from AnaSpec (San Jose, Calif.). HOAt waspurchased from Advanced ChemTech (Louisville, Ky.). NMP and DCM wereobtained from Fisher Scientific (Pittsburgh, Pa.). All other chemicalreagents for synthesis were purchased from Sigma-Aldrich (St. Louis,Mo.) and used without further purification.

Peptide synthesis and purification. All peptides were synthesizedmanually by Fmoc solid-phase methodology using DIC and HOAt as couplingreagents. When N-methylated amino acids were not commercially available,N^(α)-methylation was performed by using the optimized methodologyreported by Biron et al. (2006, J Peptide Sci 12:213-219). The followingprocedures were used for the synthesis of the linear peptides: Rinkamide MBHA resin (294 mg, 0.34 mmol/g) was placed into a 10 mL HSWpolypropylene syringe with fits on the bottom (Torviq, Niles, Mich.) andswollen in DCM (5 mL) for 30 min. After removal of the Fmoc protectinggroup (25% piperidine in NMP, 5 mL, 5 and 10 min), the resin was washedfour times with NMP (5 mL per wash) and DCM (5 mL per wash), and theindividual amino acids were coupled to the resin. For each coupling, 3equivalents (3 mmol) of the amino acid, HOAt, and DIC were used, with 10min preactivation in NMP. All couplings were performed for 1 h andmonitored by either the Kaiser test or the chloranil test. In case of apositive test result, the coupling was repeated until a negative testresult was observed.

The N-terminal amino group was acetylated with 20 equivalents of aceticanhydride and 2 equivalents of DIPEA in 5 mL of DCM for 30 min. Linearpeptides containing Cyc(Acm) residues were cyclized on resin usingthallium acetate in DMF/anisole (19:1) at ambient temperature for 3 h.The resin was washed four times with DMF, DCM, and DCM/diethylether(1:1) (each 5 mL per wash), and dried under vacuum for 4 h. The peptideswere cleaved from the resin with a mixture of 95% TFA, 2.5% water, and2.5% TIPS for 3 h. After evaporation of the TFA under vacuum, thepeptides were precipitated and washed three times with 30 mL of colddiethyl ether per wash. The liquid was separated from the solid bycentrifugation and decanted. The crude peptides were dried in air anddissolved in acetonitrile and 0.1% TFA in water (1:3) beforepurification by preparative RP-HPLC (Vydac C₁₈ 218TP152022 column,Western Analytical Products, Murrieta, Calif.) and elution with a lineargradient of 15-50% acetonitrile in aqueous 0.1% TFA solution over 35 minat a flow rate of 15 mL/min. Fractions containing the desired productswere collected, concentrated, and lyophilized. The purified peptideswere isolated in 10-15% overall yields and were >95% pure as determinedby analytical RP-HPLC (Phenomenex 00G-4041-E0 Luna 5μ C₁₈ 100A column,250×4.60 mm; Phenomenex, Torrance, Calif.). The mass of each peptide wasconfirmed using ThermoQuest Finnigan LCQ Duo and Waters MALDI micro MXinstruments.

Purification of C3. C3 was purified from fresh human plasma obtainedfrom the blood bank of the Hospital of the University of Pennsylvania.In brief, the plasma was fractionated with 15% (w/v) PEG 3350, and thepellet was resuspended in 20 mM phosphate buffer, pH 7.8, and thensubjected to anion-exchange chromatography on a DEAE-HR 40 column (50×5cm; Millipore Inc., Billerica, Mass.) with the same buffer. Proteinswere eluted with 6 L of a linear gradient (15-70%) of 20 mM phosphatebuffer, pH 7.8, containing 500 mM NaCl. C3 was further purified on asize-exclusion Superdex 200 26/60 column (Amersham Biosciences) and aMono S column (Amersham Biosciences) to separate C3 from C3(H₂O).

Inhibition of complement activation. The ability of the compstatinanalogs to inhibit the activation of the classical pathway of complementwas assessed by ELISA (Mallik et al., 2005, J Med Chem 48:274-86). Inbrief, complement was activated in human serum using an antigen-antibodycomplex in the presence or absence of compstatin analogs, and thedeposition of C3 fragments on the plate surface was detected using anHRP-conjugated polyclonal anti-C3 antibody. The absorbance data obtainedat 405 nm were translated into % inhibition, based on the absorbancecorresponding to 100% complement activation. The percent inhibition wasplotted against the peptide concentration, and the resulting data setwas fitted to the logistic dose-response function using Origin 7.0software. IC₅₀ values were obtained from the fitted parameters thatproduced the lowest χ² value. Each analog was assayed at least three toseven times. Standard deviations were all within 30% of the mean value.

ITC analysis. All ITC experiments were performed with the MicrocalVP-ITC calorimeter (Microcal Inc., Northampton, Mass.), using proteinconcentrations of 1.8-5 μM C3 in the cell and peptide concentrations of40-100 μM of individual compstatin analogs in the syringe. Alltitrations were performed in PBS (10 mM phosphate buffer with 150 mMNaCl, pH 7.4) at 25° C. using multiple peptide injections of 2-7 μLeach. The raw isotherms were corrected for the heats of dilution bysubtracting the isotherms representing peptide injections into thebuffer. The resulting isotherms were fitted to a single site of sitesmodels using Origin 7.0 software, and the model that produced the lowestχ² value was deemed to be appropriate for the respective data set. TheGibbs free energy was calculated as ΔG=ΔH−TΔS. Each experiment wasrepeated at least twice. Errors were within 20% of the mean values.

SPR analysis. The kinetics of the interaction between C3b and eachcompstatin analog was analyzed by SPR on a Biacore 3000 instrument (GEHealthcare Corp., Piscataway, N.J.) at 25° C. using PBS-T (10 mM sodiumphosphate, 150 mM NaCl, 0.005% Tween-20, pH 7.4) as the running buffer,as described above. In brief, biotinylated C3b (30 μg/mL) wasimmobilized on a streptavidin-coated sensor chip, and a two-fold serialdilution series (1 μM-500 pM) of each analog was injected for 2 min at30 μl/min, with a dissociation phase of 5-10 min. Peptide[Trp(Me)⁴]-Ac-compstatin was included in each experimental series as aninternal control and reference. Data analysis was performed usingScrubber (BioLogic Software, Campbell, Australia). The signals from anuntreated flow cell and an ensemble of buffer blank injections weresubtracted to correct for buffer effects and injection artifacts.Processed biosensor data were globally fitted to a 1:1 Langmuir bindingmodel, and the equilibrium dissociation constant (K_(D)) was calculatedfrom the equation K_(D)=k_(d)/k_(a). Peptide solutions were injected induplicate in every experiment, and each screening assay was performed atleast twice. The error of k_(a) and k_(d) were within 10% of meanvalues.

Molecular dynamics simulation. All MD simulations were performed withthe program NAMD (Phillips, et al., 2005, J. Comput. Chem. 26:1781-1802)using the CHARMM27 force field. For the free compstatin analogs, the NMRstructure (Morikis & Lambris, 2002, Biochem. Soc. Trans. 30: 1026-1036)(PDB code: 1A1P) was adopted to build starting structures. Pointmutations were introduced with the program Molecular OperatingEnvironment (MOE, Chemical Computing Group, 2005). The mutated residuesof the compstatin analogs were minimized using CHARMM (Brooks et al.,1983, J. Comput. Chem. 4: 187-217) version c33b1, with the CHARMM27(MacKerell et al., 1998, J. Phys. Chem. B 102: 3586-3616) parameter set,while harmonic constraints were placed on the backbone atoms. Theresidues of complement C3c that were missing from the crystal structurewere added using homology modeling and also minimized using CHARMM.

The crystallographic water molecules in the PDB file were maintained,and the structures were solvated in cubic periodic boxes of TIP3P(Jorgensen et al., 1983, J. Chem. Phys. 79: 926-935) water molecules.The distances between the edges of the water simulation box and theclosest atom of solutes were at least 10 Å. Sodium and chloridecounterions were then added using the VMD program (Humphrey et al.,1996, J. Mol. Graphics 14: 33-38, 27-28) in order to maintain theelectroneutrality of the systems.

The systems were first minimized in three consecutive steps, duringwhich the protein was initially held fixed and the water molecules wereallowed to move for 10,000 conjugate gradient steps; next, only theprotein backbone was held fixed for 100,000 steps; finally, all atomswere allowed to move for an additional 10,000 steps. The particle meshEwald method (Darden et al., 1993, J. Chem. Phys. 98: 10089-10092) wasused to treat long-range electrostatic interactions in periodic boundaryconditions with a grid of approximately 1 point per Å. Nonbonded van derWaals interactions were smoothly switched over 3 Å between 9 and 12 Å.Bond lengths involving bonds to hydrogen atoms were constrained by usingSHAKE (Ryckaert et al., 1977, J. Comput. Phys. 23: 327-341). The timestep for all MD simulation was 2 fs. The Nose-Hoover Langevin piston(Feller et al., 1995, J. Chem. Phys. 103: 4613-4621; Martyna et al.,1994, J. Chem. Phys. 101: 4177-4189) was used for pressure control, withthe piston period set to 200 fs and a piston decay of 100 fs. MDsimulations at 100 ps were carried out at constant volume, during whichthe systems were heated to 310 K in increments of 30 K; a subsequentisothermal isobaric MD simulation was used for 20 ns and 5 ns to adjustthe solvent density without any restraints on all the solute atoms forfree compstatin analogs and complexes, respectively. Finally, lowestenergy structures were obtained from MD-equilibrated trajectory filesand subsequently used in structure and entropy contribution analysis.

Results:

Inhibition of complement activation. A backbone N-methylation scan wasperformed on a [Tyr⁴Ala⁹]-Ac-compstatin template (peptide 1; SEQ IDNO:3) to generate analogs 2-13 (Table 1-1). Although peptide 1 was lesspotent than the current lead compound, [Trp(Me)⁴ Ala⁹]-Ac-compstatin(peptide 14, SEQ ID NO:4), it was chosen for the initial scan because ofits lower cost of synthesis. The ability of each peptide to inhibit theactivation of complement was then evaluated by ELISA and compared to theactivity of peptide 1 (Table 1-1). The most negative effect was observedfor the N-methylation of Val³, Tyr⁴ and Ala⁹, which rendered peptides 3,4, and 9 completely inactive. In contrast, N-methylation of Gly⁸ andThr¹³ produced peptides 8 and 13 with slightly increased potency (1.7-and 1.3-fold, respectively). N-methylation in all other positionsresulted in detectable, yet significantly reduced inhibitory activity(Table 1-1).

TABLE 1-1 Inhibition of classical pathway activation of complement byN^(α)-methylated analogs of [Tyr⁴ Ala⁹]-Ac-compstatin (peptide 1; SEQ ID NO: 3) Pep- IC₅₀ IC₅₀-fold tide Sequence (CP,μM)change^(a)   1^(b) Ac-I[CVYQDWGAHRC]T-NH₂ (SID 3) 2.4 1  2Ac-I[(NMe)CVYQDWGAHRC]T-NH2 7.5 0.3  3 Ac-I[C(NMe)VYQDWGAHRC]T-NH2 NA NA 4 Ac-I[CV(NMe)YQDWGAHRC]T-NH2 NA NA  5 Ac-I[CVY(NMe)QDWGAHRC]T-NH2 330.07  6 Ac-I[CVYQ(NMe)DWGAHRC]T-NH2 44 0.06  7Ac-I[CVYQD(NMe)WGAHRC]T-NH2 25 0.1  8 Ac-I[CVYQDW(NMe)GAHRC]T-NH2 1.431.7  9 Ac-I[CVYQDWG(NMe)AHRC]T-NH2 NA NA 10 Ac-I[CVYQDWGA(NMe)HRC]T-NH294 0.03 11 Ac-I[CVYQDWGAH(NMe)RC]T-NH2 32 0.08 12Ac-I[CVYQDWGAHR(NMe)C]T-NH2 154 0.02 13 Ac-I[CVYQDWGAHRC](NMe)T-NH2 1.891.3 Note: ^(a)relative to peptide 1. NA: not active, ^(b)Data from Sahuet al., 1996, J. Immunol. 157: 884-891.

We then applied the findings from the N-methylation scan to the acurrent potent analog, Ac-I[CV(^(1-Me)W)QDWGAHRC]T-NH₂ (SEQ ID NO:4;also referred to herein as [Trp(Me)⁴ Ala⁹]-Ac-compstatin, peptide 14),and synthesized analogs with selective N-methylation and amino acidsubstitutions at positions 8 and 13 (peptides 15-23; Table 1-2). Sinceprevious studies had indicated limitations for substituting the sidechain at position 8 (Morikis et al., 1998, Protein Sci. 7: 619-627;Furlong et al., 2000, Immunopharmacology 48: 199-212), modificationswere restricted to the absence (Gly⁸) or presence of N-methylation(NMeGly⁸, i.e. Sar⁸). In contrast, previous work showed that theC-terminal position 13 allowed more flexibility for substitutions andhad even suggested a preference for Ile over Thr (Morikis & Lambris,2002, Biochem. Soc. Trans. 30: 1026-1036). We therefore furtherinvestigated the importance of position 13 and designed a series of Sar⁸analogs to include various N-methylated, hydrophobic, or aromaticresidues in this position. Consistent with the results from theN-methylation scan, the introduction of a single N-methyl group atposition 8 (Sar⁸; peptide 15) increased the inhibitory potency by1.3-fold (Table 1-3). In addition, replacement of Thr by Ile at position13 led to a significant increase for both the Gly⁸ and Sar⁸ peptides.However, neither the substitution of Ile by Leu or Nle, nor theintroduction of His or Phe produced any improvement over the Ile¹³analog. In contrast, N-methylation of both Thr¹³ and Ile¹³ resulted in asignificant increase in inhibitory activity (IC₅₀=86 and 62 nM,respectively), generating the most potent compstatin analogs describedthus far.

TABLE 1-2 Evaluation of inhibitory potency, kinetic, and thermodynamicparameters for a series of compstatin analogs(Ac-Ile-c[Cys-Val-Trp(1-Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys]-Thr-NH₂)(peptide 14, SEQ ID NO: 4) with modifications in position 8 and 13.(Numbers in parentheses next to peptide numbers are SEQ ID NOs). IC₅₀k_(a) k_(d) K_(D SPR) K_(D ITC) ΔH −TΔS ΔG No. Xaa⁸ Xaa¹³ (nM) (10⁶/Ms)(10⁻³/s) (nM) (nM) (kcal/mol) (kcal/mol) (kcal/mol) 14 (4) Gly Thr 2061.0 11.3 11.9 15.0  −17.6 6.9 −10.7 15 (5) Sar Thr 159 1.3 7.2 5.5 8.5−11.7 0.6 −11.1 16 (6) Gly Ile 154 1.0 11.0 11.0 12.1  −16.6 5.7 −10.917 (7) Sar Ile 92 1.5 6.6 4.4 6.3 −14.1 2.9 −11.2 18 (8) Sar Leu 108 1.36.0 4.6 N/D N/D N/D N/D 19 (9) Sar Me 109 1.5 6.6 4.4 N/D N/D N/D N/D 20(10) Sar (NMe)Thr 86 1.3 5.1 3.9 7.2 −17.5 6.4 −11.1 21 (11) Sar(NMe)Ile 62 1.5 3.5 2.3 4.5 −17.1 5.7 −11.4 22 (12) Sar His 160 N/D N/DN/D N/D N/D N/D N/D 23 (13) Sar Phe 257 N/D N/D N/D N/D N/D N/D N/D

Binding kinetic characterization. Peptides 15-21 were furthercharacterized by SPR in order to evaluate the effect of individualsubstitutions on the kinetic profile and binding affinity for C3b (Table1-2). In general, the relative K_(D) values showed good consistency withthe ELISA results (R²=0.79; Table 1-3). N-methylation of Gly⁸ (peptides14 to 15, 16 to 17) clearly improved the binding kinetics and affinity,with significant effects on both kinetic rate constants. In contrast,the Thr-to-Ile substitutions (peptides 14 to 16, 15 to 17) had onlyslight, yet still beneficial impact on the SPR profiles. Again, thecombination of both substitutions (peptide 17) had a synergistic effect,with a 2.7-fold stronger affinity than peptide 14, as compared to theimpact of the Sar⁸ and Ile¹³ modifications alone (2.2- and 1.1-fold,respectively). Substitutions at position 13 alone appeared to primarilyinfluence the dissociation rate (k_(d)=3.4-7.2×10⁻³ s⁻¹); theassociation rate remained essentially constant for all Sar⁸ analogs(k_(a)=1.3-1.7×10⁶ M⁻¹s⁻¹). In this series, N-methylation of Thr¹³(peptide 20) and Ile¹³ (peptide 21) again had the strongest impact onthe dissociation rate, rendering analog 21 the strongest binder, with amore than 5-fold increase in affinity over peptide 14. The evaluatedisomers of Ile¹³ (Leu, Nle; peptides 18 and 19) had a negligibleinfluence on the kinetic profile and affinity, indicating a commonbinding mode for this scaffold.

Characterization of binding thermodynamics. ITC experiments wereperformed for peptides 15-17 and 20-21 in order to correlate theobserved effects on affinity and potency with their thermodynamicprofiles (Table 1-2 and 1-3). Although the absolute K_(D) values in ITCtended to be slightly higher than those from SPR, they were highlycorrelated with the ELISA and SPR results (R²=0.89 and 0.96,respectively). The highly beneficial enthalpy value (ΔH=−17.6 kcal/mol)of the previous lead compound (peptide 14) was not surpassed by any ofthe newly designed analogs. In contrast, the entire panel hadsignificantly improved entropy values (−TΔS=0.6−5.7 kcal/mol) whencompared to peptide 14 (−TΔS=6.9 kcal/mol).

TABLE 1-3 Relative improvement in the potency and binding parameters ofnewly designed compstatin analogs when compared to [Trp(Me)⁴Ala⁹]-Ac-compstatin (peptide 14) ΔΔH −TΔΔS ΔΔG No. Xaa⁸ Xaa¹³ rP rk_(a)rk_(d) rK_(D SPR) rK_(D ITC) (kcal/mol) (kcal/mol) (kcal/mol) 14 Gly Thr1.0 1.0 1.0 1.0 1.0 0  0  0  15 Sar Thr 1.3 1.3 1.6 2.2 1.8 5.9 −6.3−0.4 16 Gly Ile 1.3 1.0 1.0 1.1 1.2 1.0 −1.3 −0.2 17 Sar Ile 2.2 1.5 1.72.7 2.4 3.5 −4.1 −0.5 18 Sar Leu 1.9 1.3 1.9 2.6 N/D N/D N/D N/D 19 SarMe 1.9 1.5 1.7 2.7 N/D N/D N/D N/D 20 Sar (NMe)Thr 2.4 1.3 2.2 3.1 2.40.1 −0.5 −0.4 21 Sar (NMe)Ile 3.3 1.5 3.2 5.2 3.3 0.5 −1.3 −0.7 22 SarHis 1.3 N/D N/D N/D N/D N/D N/D N/D 23 Sar Phe 0.8 N/D N/D N/D N/D N/DN/D N/D

Peptide 15 (Sar⁸Thr¹³; −TΔS=0.6 kcal/mol) exhibited the lowest entropicpenalty of all the reported compstatin analogs. However, the majority ofthis large entropic gain was offset by a loss of favorable enthalpy(ΔΔH=5.9 kcal/mol). Similar trends were observed for the entire panel,indicating the influence of enthalpy-entropy compensation. Additionalsubstitution of Ile¹³ for Thr¹³ as in peptide 17 recaptured some of thelost enthalpy (ΔH=−14.1 kcal/mol), while yielding some of the entropygain (−TΔS=2.9 kcal/mol) in peptide 15. N-methylation in position 13, asin peptides 20-21, brought their enthalpy values even closer to that ofpeptide 14. Overall, the increased binding affinity for these peptidesappeared to be achieved mainly by a reduction in entropic penalty.Furthermore, the ITC data confirmed the SPR results indicating that itwas the Sar⁸′ and not the Ile¹³ substitution that contributed most tothe largely increased affinity of peptide 17.

MD Simulations. The large impact of even small peptide modifications onthe thermodynamic profiles of the analogs was further investigated usingMD simulations based on the NMR structure of compstatin and the crystalstructure of [Trp⁴]-Ac-compstatin with C3c (Morikis et al., 1998, supra;Janssen et al., 2007, J. Biol. Chem. 282: 29241-29247). In the case ofN-methylation at position 8 (peptide 17), we suspected that thismodification affected the side chain of the critical residue Trp⁷, whichis directly connected to the methylated Gly⁸ nitrogen and occupies atight pocket. MD simulations were therefore performed to compare thedistribution of water molecules in the Trp⁷ binding pockets of peptides14 and 17. We found that whereas four water molecules could be observedfor peptide 14, none were found after repeating the simulation withpeptide 17. This result indicates that N-methylation at position 8allows the side chain of Trp⁷ to better fit into the C3c binding pocket.

Previous comparisons between the solution-based and protein-boundstructures have revealed significant conformational rearrangement,including a shift in the important β-turn (Janssen et al., 2007, supra).Since N-methylation has been reported to affect the local conformationof the peptide backbone, we performed MD simulations for peptide 14 and17 in the absence and presence of C3c and then compared the resultinglowest energy conformers of the free and bound peptides (Chatterjee etal., 2008, Acc. Chem. Res. 41: 1331-1342). The results showed that theβ-turn encompassing residues 5-8 opened, and a new turn was formedbetween residues 8 and 11 in the free structures of both peptides. Also,the β-turns overlaid well with those in the bound structures. However,an intramolecular hydrogen bond between Trp⁷ and Arg¹¹ with a distanceof 2.9 Å was formed only in the case of peptide 17, likely constrainingthe conformation of free 17 and making it more rigid.

EXAMPLE 2

This example describes an improved synthesis, and plasma half-lifedetermination, of a compstatin analog (peptide 17 described in Example1: Ac-Ile-c[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His-Arg-Cys]-Ile-NH₂;SEQ ID NO:7) conjugated to an albumin-binding peptide (ABP) or analbumin-binding small molecule (ABM), shown below.

ABP: Ac-RLIEDICLPRWGCLWEDD-NH₂ (C-C disulfide bond) (SEQ ID NO:14)

Two mini-PEG-3 molecules were used as a spacer and coupled to theC-terminal of peptide 17.

For comparison, the plasma half-life of the unconjugated peptide 17 wasalso determined.

Materials and Methods:

Abbreviations. Ac, acetyl group; Acm, acetamidomethyl; Acm,acetamidomethyl; DCM, dichloromethane; DIC, 1,3-diisopropylcarbodiimide;DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethyl-formamide; ELISA,enzyme-linked immunosorbent assay; ESI, electrospray ionization; Fmoc,9-fluorenylmethoxycarbonyl; HLB, hydrophilic-lipophilic balanced; HOAt,1-hydroxy-7-aza-benzotriazole; HSW, Henke Sass Wolf; ITC, isothermaltitration calorimetry; MALDI, matrix-assisted laser desorptionionization; MBHA, 4-methylbenz-hydrylamine; Mmt, Monomethoxytrityl;NanoESI, nanoelectrospray ionization; NMP, N-methylpyrrolidinone; PyBOP,benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; SPR,surface plasmon resonance; TBTA, Tris-(benzyltriazolylmethyl)amine; TEA,triethylamine; TFA, trifluoroacetic acid; TIPS, triisopropylsilane; Trt,trityl.

Materials. DIC and Fmoc-Trp(Me)-OH were purchased from AnaSpec (SanJose, Calif.). Low-loading NovaSyn® TGR resin and other Fmoc-amino acidswere obtained from Novabiochem (San Diego, Calif.). Mini-PEG andmini-PEG-3 were purchased from Peptide International (Louisville, Ky.).HOAt was purchased from Advanced ChemTech (Louisville, Ky.). ABM wasobtained from Enamine Ltd. (Kiev, Ukraine). NMP and DCM were obtainedfrom Fisher Scientific (Pittsburgh, Pa.). Water was purified using aMilli-Q water purification system (Millipore Corporate, Billerica,Mass.). All other chemical reagents for synthesis were purchased fromSigma-Aldrich (St. Louis, Mo.) and used without further purification.

Synthesis of linear peptides (peptide 17-mini-(PEG-3)₂-Lys(Mmt)-NH₂ andABP). All peptides were synthesized manually by Fmoc solid-phasemethodology using DIC and HOAt as coupling reagents. In brief, resin(294 mg, 0.34 mmol/g) was placed into a 10 mL HSW polypropylene syringewith frits on the bottom (Torviq, Niles, Mich.) and swollen in DCM (5mL) for 30 min. After removal of the Fmoc protecting group (25%piperidine in NMP, 5 mL, 5 and 10 min), the resin was washed four timeswith NMP (5 mL per wash) and DCM (5 mL per wash), and the individualamino acids were coupled to the resin. For each coupling, 3 equivalents(0.3 mmol) of the amino acid, HOAt, and DIC were used, with 10 minpreactivation in NMP. All couplings were performed for 1 h and monitoredto completion by either the Kaiser test or the chloranil test. Ifnecessary, the N-terminal amino group was acetylated with 20 equivalentsof acetic anhydride and 2 equivalents of DIPEA in 5 mL of DCM for 30min.

Peptide cyclization, modification and cleavage. Linear peptidescontaining Cyc(Acm) residues were cyclized on-resin using 1.2 equivalentof thallium trifluoroacetate in DMF/anisole (19:1) at ambienttemperature for 3 h. To synthesize azido-peptide 17, the side chain Mmtprotecting group of the C-terminal Lys of the peptide 17 derivative wasremoved using 1% TFA in DCM with 5% TIPS. Then, 2-azidoacetic acid wascoupled to the side chain using PyBOP/HOAt/DIPEA in NMP. Peptide 17-ABMwas synthesized in similar way. To synthesize Alkyne-ABP, propiolic acidwas coupled to the N-terminal of ABP using DIC/HOAt in NMP/DCM (1:1).Resin was thoroughly washed with DCM, DCM/diethylether (1:1), and driedunder high vacuum for 4 h before the peptides were cleaved in a mixtureof 95% TFA, 2.5% water, and 2.5% TIPS for 2 h. After evaporation of theTFA under vacuum, the peptides were precipitated and washed three timeswith 30 mL of cold diethyl ether per wash. The liquid was separated fromthe solid by centrifugation and decanted. The crude peptides were driedin air and dissolved in acetonitrile and 0.1% TFA in water (1:3) forHPLC purification.

Cupper(I) mediated azide-alkyne Huisgen cycloaddition for the synthesisof peptide 17-ABP. 50 mg (22 μmol) of each purified azide and alkynepeptide was dissolved in 5 mL of t-BuOH/H₂O (2:1). 10 equiv (220 μmol)of TEA was added to make the solution basic. Then 5% (1.1 μmol) ofCuSO₄, 25% of sodium ascorbic acid, and 1% of TBTA was added to themixture. The mixture was stirred overnight, monitored by HPLC-MS. It wasthen concentrated under vacuum and purified by reverse phase HPLC.

Peptide purification. The peptides were injected into a preparativeRP-HPLC column (Xbridge™ BEH130 Prep C18 5 μm 19×150 mm, PN #186003945,Waters, Milford, Mass.) and eluted with a linear gradient of 15-50%acetonitrile in 0.1% TFA over 15 min at a flow rate of 20 mL/min.Fractions containing the desired products were collected baseD on mass,and lyophilized. The purified peptides were >95% pure as determined byanalytical RP-HPLC (Xbridge™ BEH130 C18 5 μm, 4.6×150 mm, PN #186003580,Waters, Milford, Mass.). The mass of each peptide was confirmed usingWaters MALDI micro MX instruments or SYNAPT HDMS.

Inhibition of complement activation. The ability of the compstatinanalogs to inhibit the activation of the classical pathway of complementwas assessed by ELISA as described in Example 1. Each conjugate wasassayed at least three times.

SPR analysis. The kinetics of the interaction between C3b and eachcompstatin analog was analyzed by SPR on a Biacore 3000 instrument (GEHealthcare Corp., Piscataway, N.J.) at 25° C. using PBS-T (10 mM sodiumphosphate, 150 mM NaCl,0.005% Tween-20, pH 7.4) as the running buffer,as described in Example 1. In brief, biotinylated C3b (30 μg/ml) wasimmobilized on a streptavidin-coated sensor chip, and a two-fold serialdilution series (1 μM-500 pM) of each analog was injected for 2 min at30 μl/min, with a dissociation phase of 5-10 min. Peptide 17(unconjugated) was included in each experimental series as an internalcontrol and reference. Data analysis was performed using Scrubber(BioLogic Software, Campbell, Australia). The signals from an untreatedflow cell and an ensemble of buffer blank injections were subtracted tocorrect for buffer effects and injection artifacts. Processed biosensordata were globally fitted to a 1:1 Langmuir binding model, and theequilibrium dissociation constant (K_(D)) was calculated from theequation K_(D)=k_(d)/k_(a). Peptide solutions were injected in duplicatein every experiment, and each screening assay was performed at leasttwice.

Extraction of compstatin analogs from plasma samples by SPE. A 96-wellplate HLB Oasis 10 mg (Waters, Milford, Mass.) was employed forextraction. The SPE material was conditioned by addition of 500 μl ofmethanol followed by addition of 500 μL of milli-Q water. Sample wasprepared by addition of the internal standard followed by 1:1 dilutionwith 4% H₃PO₄. After loading the sample, washing was carried out with500 μL of 5% methanol in 0.1% formic acid. Sample was eluted with 150 μLof 65% methanol in 0.1% formic acid and collected in the collectionplate. Solvent was evaporated to dryness in a speed-vac concentrator andreconstituted in 5% acetonitrile in 0.1% formic acid. Samples were keptat −20° C. until analysis.

Isolation of peptide 17-ABP and peptide 17-ABM from plasma samples bydigestion. Baboon plasma samples, 40 μL, were mixed with internalstandard and dissolved 1:1 with 40 mM ammonium carbonate buffer.Rapigest detergent was added to a final concentration of 0.1%. Disulfidebridges were reduced in 5 mM DTT for 30 min at 60° C. Alkylation ofcysteines was done by addition of iodoacetamide to a final concentrationof 15 mM and incubation for 30 min in dark. The sample was enzymaticallydigested by addition of 16 μL of a 1 μG/μL trypsin solution andincubation overnight at 37° C. After that, the sample pH was loweredwith 5% TFA to induce detergent degradation. To avoid nonspecificadsorption of very hydrophobic peptides, acetonitrile was added to 20%.The samples were centrifuged at 6° C. and 14000 rpm for 30 min and thesupernatant was diluted with 0.1% formic acid to reduce acetonitrileconcentration to 10% prior to filtration with a 10 kDa cut-off microconcentrifugal filter (Millipore, Billerica, Mass.). The filter was washedwith 50 μL of 10% ACN in 0.1% formic acid and the collected sample wasevaporated to dryness and reconstituted with 10% ACN in 0.1% formicacid.

LC-MS/MS analysis. LC-MS/MS analysis was performed on a SYNAPT HDMS(Waters, Milford, Mass.) controlled by MassLynx 4.1 software (Waters)and equipped with a nanoESI source. Each sample was injected intriplicate. A nanoACQUITY UPLC (Waters) system was used for peptideseparation by reversed-phase liquid chromatography. After injection,analytes were trapped for 3 min with 3% mobile phase A (0.1% formic acidin water) at 5 μl/min on a 5 μm Symmetry C18 column (180 μm×20 mm,Waters) and further separated on a 1.7 μm BEH130 C18 column (75 μm×150mm, Waters). The analytical column temperature was held at 35° C.Peptides were separated at flow rate 0.3 μl/min. The gradient was linear3-40% B (0.1% formic acid in acetonitrile), either 50 min long, or 60min for the digested samples. The capillary voltage was 3.2 kV, the conevoltage was 35 V and the source temperature was 100° C.[Glu1]-fibrinogen peptide was used for lock-mass correction with asampling rate of 30 s. Mass spectra were acquired in positive mode overan m/z range 400-2000 Da at scan rate 0.6 s. The time window used forthe MS/MS function was ±3 min of the retention time of the selectedpeptide. The presence of the analyte was confirmed by MS/MS. Selectivitywas studied by analysis of blank samples to determine the presence ofany interference coeluting with the analyte.

In vivo retention. Juvenile baboons (P. Anubis, Baboon ResearchResources, University of Oklahoma) weighing 5-8 kg were used. Threebaboons were used for the study; one for each compound. All animalsreceived a bolus dose of peptides (10 mg) by injection through theperipheral vein. Blood samples for the LC-MS/MS assay were collected in1-ml plastic tubers containing 50 μg lepirudin and centrifuged at 2000 gfor 20 min at 4° C. for plasma separation. Plasma samples were stored at−70° C. Blood samples were collected at 20, 40, 60 90, 120 min afterinjection of peptide 17; and 1 min, 30 min then 1, 6, 24 and 48 hrsafter injection of peptide 17-ABP and peptide 17-ABM.

Results:

Synthesis of peptide 17-ABM. Peptide 17-ABM was obtained after solidphase peptide synthesis and HPLC purification, as summarized in thereaction scheme below. The linear peptide was synthesized with a singlecoupling of each amino acid. Both thallium trifluoroacetate and iodinewas evaluated for the disulfide bond formation. The former yieldedcleaner reactions and was thereafter used for all cyclizations. The massof peptide 17-ABM was confirmed by HPLC-MS and ESI-TOF ([MH]²⁺ calc.1211.06, found 1211.05).

Synthesis of peptide 17-ABP. In solution azide-alkyne Huisgencycloaddition was used for the conjugation, according to the reactionscheme below. The 2-azidoacetic acid was synthesized from 2-bromoaceticacid and sodium azide. It was then coupled to the C-terminal Lys sidechain after formation of the disulfide bond on resin. Intermediates 2and 3 were obtained in 12.7% and 12.3% yield, respectively, aftercleavage and HPLC purification.

Peptide 17-ABP (“4” in the scheme above)

Three different solvent systems were compared for the azide-alkyneHuisgen cycloaddition. The best result was observed with t-BuOH/H₂Osystem, followed by ACN/H₂O system. No product was observed when DMFalone was used as solvent. The importance of tertiary base was alsoevaluated. No product was detected after 2 h without addition of excessTEA. Under optimized conditions, the reaction was clean and peptide17-ABP was isolated in 50% yield after HPLC purification. The mass ofthe product was further confirmed by ESI-TOF ([MH]⁴⁺ calc. 1131.78,found 1131.52).

Inhibition of complement activation. The ability of peptide17-ABM andpeptide 17-ABP to inhibit classical pathway complement activation wasevaluated by ELISA, using human serum. The results are shown in Table2-1.

TABLE 2-1 Results of ELISA and SPR analyses of peptide 17 and ABP or ABMconjugates IC₅₀ K_(on) IC₅₀ (fold (10⁶, K_(off) K_(D) Peptide (nM, CP)change*) M⁻¹ · s⁻¹) (10⁻³, s⁻¹) (nM) Peptide 17 92 1 1.0 6.6 4.4 Peptide17-ABM 137 0.67 1.2 5.6 4.7 Peptide 17-ABP 242 0.38 0.1 3.6 32 *Foldchange is relative to peptide 17

Plasma concentration in baboons. The plasma concentrations of peptide 17and the ABP and ABM conjugates were determined using LC-MS/MS after anintravenous bolus injection into baboons. Peptide 17 showed a half-lifeof around 60 min. Peptide 17-ABM displayed a 5-fold improvement with ahalf-life of 5 h. The longest half-life of 21 h was observed for peptide17-ABP, which was 21-fold greater than that of unconjugated peptide 17.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

What is claimed:
 1. A compound comprising a modified compstatin peptide(ICVVQDWGHHRCT (cyclic C2-C12); SEQ ID NO:1) or analog thereof, in whichthe Gly at position 8 is modified to constrain the backbone conformationof the peptide at that location.
 2. The compound of claim 1, wherein thebackbone is constrained by replacing the Gly with N-methyl Gly.
 3. Thecompound of claim 2, further comprising replacement of His at position 9with Ala.
 4. The compound of claim 3, further comprising replacement ofVal at position 4 with Trp or an analog of Trp.
 5. The compound of claim4, wherein the analog of Trp at position 4 is 1-methyl Trp or 1-formylTrp.
 6. The compound of claim 4, further comprising replacement of Trpat position 7 with an analog of Trp.
 7. The compound of claim 6, whereinthe analog of Trp at position 7 is a halogenated Trp.
 8. The compound ofclaim 3, further comprising acetylation of the N-terminal residue. 9.The compound of claim 1, further comprising replacing the Thr atposition 13 with Ile, Leu, Nle, N-methyl Thr or N-methyl Ile.
 10. Thecompound of claim 1, which is a compstatin analog comprising a peptidehaving a sequence of SEQ ID NO:2, which is:Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa3-Gly-Xaa4-His-Arg-Cys-Xaa5 (cyclic C2-C12)in which Gly at position 8 is modified to constrain the backboneconformation at that location; wherein: Xaa1 is Ile, Val, Leu, Ac-Ile,Ac-Val, Ac-Leu or a dipeptide comprising Gly-Ile; Xaa2 is Trp or ananalog of Trp, wherein the analog of Trp has increased hydrophobiccharacter as compared with Trp; Xaa3 is Trp or an analog of Trpcomprising a chemical modification to its indole ring wherein thechemical modification increases the hydrogen bond potential of theindole ring; Xaa4 is His, Ala, Phe or Trp; and Xaa5 is Thr, Ile, Leu,Nle, N-methyl Thr or N-methyl Ile, wherein a carboxy terminal —OH of anyof the Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile optionally isreplaced by —NH₂.
 11. The compound of claim 10, wherein the Gly atposition 8 is N-methylated, and Xaa1 is Ac-Ile, Xaa2 is 1-methyl-Trp or1-formyl-Trp, Xaa3 is Trp, Xaa4 is Ala, and Xaa5 is Thr, Ile, Leu, Nle,N-methyl Thr or N-methyl Ile.
 12. The compound of claim 11, wherein Xaa5is Ile, N-methyl Thr or N-methyl Ile.
 13. The compound of claim 11,which comprises any of SEQ ID NOS: 5, 7, 8, 9, 10 or
 11. 14. Thecompound of claim 1, further comprising an additional component thatextends the in vivo retention of the compound.
 15. The compound of claim14, wherein the additional component is polyethylene glycol (PEG). 16.The compound of claim 14, wherein the additional component is an albuminbinding small molecule.
 17. The compound of claim 14, wherein theadditional component is an albumin binding peptide.
 18. The compound ofclaim 15, wherein the albumin binding peptide comprises the sequenceRLIEDICLPRWGCLWEDD (SEQ ID NO: 14).
 19. The compound of claim 15,comprising any one of SEQ ID NOS: 5, 7, 8, 9, 10 or 11 linked to thealbumin binding peptide.
 20. The compound of claim 15, wherein thecompound and the albumin binding peptide are separated by a spacer. 21.The compound of claim 20, wherein the spacer is a polyethylene glycolmolecule.
 22. A pharmaceutical composition comprising the compound ofclaim 1 and a pharmaceutically acceptable carrier.